Dengue fever epidemics have increased over the last decades and disease incidence has risen 30-fold in the last 50 years. The viruses are now endemic in more than 100 countries and over 200 million infections occur annually with >50% of the world's population at risk. This rise is fueled in part by tremendous population growth, a shif in demographics towards urbanization and increased international trade and travel. The lack of approved dengue-specific prophylactic or therapeutic drugs and vaccines makes urgent the need for new, cost- effective and efficacious disease-control tools that are safe for people and the environment. This need justifies efforts to develop genetic approaches for controlling virus transmission. Long-term, sustainable genetic control will require the deployment of strategies designed to be resilient to the immigration of susceptible mosquitoes and dengue-infected people. Strains for population modification have the appropriate performance features for this purpose. Wild mosquitoes immigrating into a region populated by engineered, virus-resistant mosquitoes will acquire the gene by mating with the local insects, and persons with dengue moving into the same region will not be able to infect the resident vectors, and therefore are not a source for infection of other people. We will exploit the molecular mechanisms of the CRISPR/Cas9 biology to develop an autonomous nicking endonuclease-mediated gene drive (NMG) system for site-specific, transgene copy number amplification in the mosquito germline. The working hypothesis is that it will be possible to engineer a Cas9 nicking endonuclease to increase transgene frequency at each generation in the major dengue-vector mosquito Aedes aegypti. Transgene inheritance frequencies that exceed Mendelian expectations will demonstrate the proof-of-principle for the NMG system. Towards these ends, our Specific Aims are: 1) synthesize and validate site-specific endonuclease activity of an adaptable NMG system targeting the wild-type Ae. aegypti kynurenine hydroxylase (kh+) gene and 2) analyze transgenic lines for evidence of autonomous gene-drive properties by measuring inheritance patterns of NMG transgenes. The successful development of the NMG system will have a profound impact on the further testing and possible final adoption of a population-replacement strategy for controlling dengue. The innovative design of the NMG system meets a number of stringent criteria for genetic- control technologies including male-only releases, no horizontal transfer potential to other species and maintenance of linkage of anti-viral effector genes with the drive system. It also provides the ability to target specifically any region in the mosquito genome and introduce additional effector genes by changing the targeted gene sequence or exploiting recombinase-mediated insertion at a 'docking' site.